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pH Dependence of oxy and deoxy cobalt-substituted leghemoglobin from soybean

An electron spin resonance study

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Abstract

In leghemoglobin a, which is the major hemoglobin component in soybean root nodules, the haem iron has been replaced by cobalt. The electron spin resonance (ESR) of frozen solutions of the cobalt-substituted leghemoglobin has been studied at 77 K in the deoxy and oxy forms respectively. Both ligation states exhibit rhombic g tensors. The hyperfine constants of 59Co, 14N-imidazole (residue of the proximal histidine) and 14N-pyrroles are determined for the three principal directions of the g tensor. Both, the oxy and the deoxy state exhibit pH-dependent changes of the hyperfine structures. For oxy cobalt leghemoglobin a quantitative analysis of the pH titration and of the ESR parameters of the low and high-pH forms respectively are performed. The interconversion of the low and the high-pH forms is controlled by a proton-dissociating group with pK=6.4 which is most probably the distal histidine. g tensors and hyperfine constants are compared with those described for oxy cobalt myoglobin crystal spectra [34] allowing assignments of the low and high-pH species of leghemoglobin to stereoelectronic structures with non-equivalent and equivalent dioxygen atoms respectively. Hydrogen-bonding of the distal histidine with dioxygen favours the structure with equivalent oxygen atoms. The pH dependence of the deoxy form is interpreted as interaction of the proximal imidazole with the central cobalt atom.

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References

  1. Wittenberg JB, Bergersen FJ, Appleby CA, Turner GC (1974) Facilitated oxygen diffusion. The role of leghemoglobin in nitrogen fixation by bacteroids isolated from soybean root nodules. J Biol Chem 249:4057–4066

    Google Scholar 

  2. Ellfolk N (1960) Crystalline leghemoglobin. I. Purification procedure. Acta Chem Scand 14:609–616

    Google Scholar 

  3. Broughton WJ, Dilworth MJ (1972) Amino acid composition and relationships of lupin and Serradella leghemoglobins. Biochim Biophys Acta 317:266–276

    Google Scholar 

  4. Peive YV, Atausov BP, Zhiznevskaya GY, Krasnobaeva NN (1972). Dokl Acad Nauk SSSR 202:482–485

    Google Scholar 

  5. Vainshtein BK, Amtyunyan EG, Kuranova IP, Borisov VV, Sosfenov NI, Pavlovskii AG, Grebenko AI, Konareva NV (1974). Dokl Acad Nauk SSSR 216:690–693

    Google Scholar 

  6. Richardson M, Dilworth MJ, Scawen MD (1975) The amino acid sequence of leghemoglobin I from root nodules of broadbean (Vicia faba L.). FEBS Lett 51: 33–37

    Google Scholar 

  7. Lehtovaara P, Ellfolk N (1974) The primary structure of kidney bean leghemoglobin. FEBS Lett 43:239–240

    Google Scholar 

  8. Ellfolk N, Sievers G (1971) The primary structure of soybean leghemoglobin. Acta Chem Scand 25:3532–3534

    Google Scholar 

  9. Ellfolk N (1972) LeghÄmoglobin, ein PflanzenhÄmoglobin. Endeavour 31:139–142

    Google Scholar 

  10. Appleby CA, Blumberg WE, Peisach J, Wittenberg BA, Wittenberg JB (1976) Leghemoglobin. An electron paramagnetic resonance and optical spectral study of the free protein and its complexes with nicotinate and acetate. J Biol Chem 251:6090–6096

    Google Scholar 

  11. Appleby CA (1962) The oxygen equilibrium of leghemoglobin. Biochim Biophys Acta 60:226–235

    Google Scholar 

  12. Imamura T, Riggs A, Gibson QH (1972) Equilibria and kinetics of ligand binding by leghemoglobin from soybean root nodules. J Biol Chem 247:521–526

    Google Scholar 

  13. Appleby CA (1974) In: Quispel A (ed) The biology of nitrogen fixation. North Holland, Amsterdam, pp 521–544

    Google Scholar 

  14. Appleby CA, Wittenberg BA, Wittenberg JB (1973) Nicotinic acid as a ligand affecting leghemoglobin structure and oxygen reactivity. Proc Natl Acad Sci USA 70:564–568

    Google Scholar 

  15. Ellfolk N (1961) Crystalline leghemoglobin. IV. Spectroscopic studies of the two main met leghemoglobin components and some of their fatty acid complexes. Acta Chem Scand 15:975–984

    Google Scholar 

  16. Ikeda-Saito M, Yamamoto H, Imai K, Kayne FJ, Yonetani T (1977) Studies on cobalt myoglobins and hemoglobins. Preparation of isolated chains containing cobaltous protoporphyrin IX and characterization of their equilibrium and kinetic properties of oxygenation and EPR spectra. J Biol Chem 252:620–624

    Google Scholar 

  17. Ikeda-Saito M, Yamamoto H, Yonetani T (1977) Studies on cobalt myoglobins and hemoglobins. Electron paramagnetic resonance of iron cobalt hybrid hemoglobins and its implication for the heme-heme interaction and for the alkaline Bohr-effect. J Biol Chem 252:8639–8644

    Google Scholar 

  18. Yonetani T, Yamamoto H, Iizuka T (1974) Studies on cobalt myoglobins and hemoglobins. III. Electron paramagnetic resonance studies of reversible oxygenation of cobalt myoglobins and hemoglobins. J Biol Chem 249:2168–2174

    Google Scholar 

  19. Dickinson LC, Chien JCW (1973) Electron paramagnetic resonance of single crystal deoxycobalt-hemoglobin. Biochem Biophys Res Commun 51:587–592

    Google Scholar 

  20. Ikeda-Saito M, Brunori M, Yonetani T (1980) Oxygenation and EPR spectral properties of Aplysia myoglobins containing cobaltous prophyrins. Biochim Biophys Acta 533:173–180

    Google Scholar 

  21. Ikeda-Saito M, Iizuka T, Yamamoto H, Kayne FJ, Yonetani T (1977) Studies on cobalt myoglobins and hemoglobins. Interaction of sperm whale myoglobin and Glycera hemoglobins with molecular oxygen. J Biol Chem 252:4882–4887

    Google Scholar 

  22. Chien JCW, Dickinson LC (1972) Electron paramagnetic resonance of single crystal oxycobaltmyoglobin and deoxycobaltmyoglobin. Proc Natl Acad Sci USA 69:2783–2787

    Google Scholar 

  23. Gersonde K, Twilfer H, Overkamp M (1978) ESR and oxygen-binding properties of monomeric allosteric cobalt haemoglobins. Presented at the VIIIth International Conference on Magnetic Resonance in Biological Systems, Nara, Japan, Abstracts E10:106

    Google Scholar 

  24. Christahl M, Gersonde K, Raap A, Appleby C (1979) Electron spin resonance study of cobalt leghaemoglobin. Presented at the Annual Meeting of the Deutsche Gesellschaft für Biophysik, Konstanz, Germany, Abstracts A14:15

    Google Scholar 

  25. Wittenberg JB, Appleby CA, Wittenberg BA (1972) The kinetics of the reactions of leghemoglobin with oxygen and carbon monoxide. J Biol Chem 247:527–531

    Google Scholar 

  26. Teale FWJ: Cleavage of the haem-protein link by acid methylethyl ketone. Biochim Biophys Acta 35:543

  27. Yonetani T, Yamamoto H, Woodrow III GV (1974) Studies on cobalt-myoglobins and hemoglobins. I. Preparation and optical properties of myoglobins and hemoglobins containing cobalt proto-, meso-, and deuteroporphyrins and thermodynamic characterization of their reversible oxygenation. J Biol Chem 249:682–690

    Google Scholar 

  28. Twilfer H, Gersonde K, Christahl M (1981) Resolution enhancement of EPR spectra using the Fourier transform technique. Analysis of nitrosyl cytochrome c oxidase in frozen solution. J Magn Reson

  29. Tovros BS, Kitko DJ, Drago RS (1975) Nature of the bound O2 in a series of cobalt dioxygen adducts. J Am Chem Soc 98:5144–5152

    Google Scholar 

  30. Drago RS (1979) The spin-pairing model for the binding of dioxygen to transition metal complexes. In: ESR and NMR of paramagnetic species in biological and related systems. Proceedings of the NATO Adv. Study Inst., pp 289–301

  31. Melamud E, Silvers BL, Dori Z (1974) Electron paramagnetic resonance of mononuclear cobalt oxygen carriers labelled with oxygen-17. J Am Chem Soc 96:4689–4690

    Google Scholar 

  32. Getz D, Melamud E, Silver BL, Dori Z (1975) Electronic structure of dioxygen in cobalt(II) oxygen carriers, singlet oxygen or O −2 ?. J Am Chem Soc 97:3846–3847

    Google Scholar 

  33. Fantucci P, Valenti V (1976) Molecular orbital study of a cobalt reversible oxygen carrier. J Am Chem Soc 98:3832–3838

    Google Scholar 

  34. Dickinson C, Chien J (1980) Electron paramagnetic resonance crystallography of 17O-enriched oxycobaltomyoglobin: Stereoelectronic structure of the cobalt dioxygen system. Proc Natl Acad Sci USA 77:1235–1239

    Google Scholar 

  35. Pauling L (1964) Nature of the iron-oxygen bond in oxy haemoglobin. Nature 203:182–183

    Google Scholar 

  36. Griffith JS (1956) On the magnetic properties of some haemoglobin complexes. Proc R Soc (Lond) Ser A 235:23–36

    Google Scholar 

  37. Collman JP, Gagne RR, Reed CA, Robinson WT, Rodley GA (1974) Structure of an iron (II) dioxygen complex; a model for oxygen carrying heme proteins. Proc Natl Acad Sci USA 71:1326–1329

    Google Scholar 

  38. Petsko GA, Rose D, Tseroglou D, Ikeda-Saito M, Yonetani T (1978) The structure of oxy cobalt myoglobin at 1.5 å resolution. In: Dutton PL, Leigh JS Jr, Scarpa A (eds) Frontiers of biological energetics. From electrons to tissues. Academic Press, New York, pp 1011–1017

    Google Scholar 

  39. Johnson RN, Bradbury JH, Appleby CA (1978) A proton magnetic resonance study of the distal histidine of soybean leghemoglobin. J Biol Chem 253:2148–2154

    Google Scholar 

  40. Appleby CA (1969) Properties of leghemoglobin in vivo, and its isolation as ferrous oxyleghemoglobin. Biochim Biophys Acta 188:222–229

    Google Scholar 

  41. La Mar GN, Budd DL, Sick H, Gersonde K (1978) Acid Bohr effects in myoglobin characterized by proton NMR hyperfine shifts and oxygen binding studies. Biochim Biophys Acta 537:270–283

    Google Scholar 

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Authors and Affiliations

  1. Abteilung Physiologische Chemie, Rheinisch-WestfÄlische Technische Hochschule Aachen, Schneebergweg 213, D-5100, Aachen, Federal Republic of Germany

    M. Christahl, A. Raap & K. Gersonde

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  1. M. Christahl
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  2. A. Raap
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  3. K. Gersonde
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Christahl, M., Raap, A. & Gersonde, K. pH Dependence of oxy and deoxy cobalt-substituted leghemoglobin from soybean. Biophys. Struct. Mechanism 7, 171–186 (1981). https://doi.org/10.1007/BF00539177

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  • DOI: https://doi.org/10.1007/BF00539177

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